Methods of electrochemically measuring an analyte with a test sequence having a pulsed dc block as well as devices, apparatuses and systems incorporating the same

ABSTRACT

Methods are disclosed for measuring an analyte concentration in a fluidic sample. Such methods further allow one to correct and/or compensate for confounding variables such as hematocrit (Hct), temperature or both before providing an analyte concentration. The measurement methods utilize information obtained from test sequences having at least one AC block and at least one pulsed DC block, where pulsed DC block includes at least one recovery potential, and where a closed circuit condition of the electrode system is maintained during the DC block. Also disclosed are devices, apparatuses and systems incorporating the various measurement methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of U.S. application Ser. No.14/851,621 (filed 11 Sep. 2015, issuing as U.S. Pat. No. 10,119,931 on 6Nov. 2018), which further claims the benefit of Intl Patent ApplicationNo. PCT/EP2014/054965 (filed 13 Mar. 2014), which further claims thebenefit of US Provisional Patent Application Nos. 61/792,748 (filed 15Mar. 2013) and 61/801,826 (filed 15 Mar. 2013), each of which isincorporated herein by reference as if set forth in its entirety.

TECHNICAL FIELD

The disclosure relates generally to mathematics and medicine, and moreparticularly, it relates to methods of electrochemically measuring ananalyte in a fluidic sample based upon response currents obtained froman electrical test sequence having at least one pulsed direct current(DC) block.

BACKGROUND

Significant benefits can be realized from electrochemically measuringanalytes in fluidic samples (i.e., biological or environmental). Forexample, individuals with diabetes can benefit from measuring glucose.Those potentially at-risk for heart disease can benefit from measuringcholesterols and triglycerides among other analytes. These are but a fewexamples of the benefits of measuring analytes in biological samples.Advancements in the medical sciences are identifying a growing number ofanalytes that can be electrochemically analyzed in a fluidic sample.

The accuracy of present approaches to electrochemically measuringanalytes such as glucose can be negatively affected by a number ofconfounding variables including variations in reagent thickness, wettingof the reagent, rate of sample diffusion, hematocrit (Hct), temperature,salt and other confounding variables. These confounding variables cancause an increase or decrease in an observed magnitude of, for example,a current response that is proportional to glucose, thereby causing adeviation from the “true” glucose concentration.

A number of approaches are known for correcting errors attributable tovariation in test sample and biosensor characteristics. Some approachesseek to perform active correction on analyte measurements. For example,US Patent Application Publication No. 2009/0236237 discloses biosensormeasurement systems including temperature correction algorithms forcorrecting analyte measurements based upon measurements of the ambienttemperature, the temperature of the biosensor itself, or the timebetween when a biosensor is attached to a measurement device and asample is provided to the biosensor. In another example, U.S. Pat. No.7,407,811 discloses a system and method for measuring blood glucose thatcorrects for variation in temperature, Hct and other confoundingvariables by measuring impedance of a blood sample to an AC excitationand using the impedance (or impedance derived admittance and phaseinformation) to correct for the effects of such interferents.

Other approaches seek to control the physical characteristics ofbiosensors. For example, U.S. Pat. No. 7,749,437 discloses methods forcontrolling reagent thickness and uniformity.

There also have been attempts to perform electrochemical analytemeasurements using pulsed signals. For example, Barker et al. disclosethat an alternating polarographic square wave potential can be appliedto a test cell and that the amplitude of the AC current response wasmeasured just before each change of applied voltage to detectconcentrations of metallic ions. See, Barker et al. (1952) Int'l Congr.Anal. Chem. 77:685-696 Barker et al. also disclose that their methodameliorates the undesirable effect of the rate of double-layercapacitance current on the sensitivity of an A/C polarograph.

In addition, Gunasingham et al. disclose a pulsed amperometric detectionmethod for a mediator-based enzyme electrode that applies a potentialpulse signal to a working electrode and measures a current response.See, Gunasingham et al. (1990) J. Electroanal. Chem. 287:349-362. Thepulse signal alternates between a base potential and an excitationpotential. Excitation potential pulse durations ranging from less than100 msec to more than 1 sec are disclosed, and current sampling occurredduring the last 16.7 msec of each oxidation potential pulse. See also,U.S. Pat. No. 5,312,590, which discloses applying a pulsed excitationsequence that is alternated between 0 V for 300-500 msec and 150 mV for50-60 msec.

Furthermore, Champagne et al. disclose a voltammetric measurement methodthat applies variable potential signals to electrodes of anelectrochemical cell to produce an electrochemical reaction and measuresthe resulting current response. See, U.S. Pat. No. 5,980,708. A squarewaveform potential was used to drive test cell electrodes. Positive andnegative current responses were integrated over time periods within acurrent response, and the integrated currents were summed to calculate acurrent measurement. An example of a pulsed voltammetric measurementmethod disclosed therein used an input signal having a pulse height of30 mV, a step height of 5 mV, a cycle period of 100 msec, a pulse widthof 40 msec and a sample time of 35 msec. Champagne et al. furtherdisclose ensuring that the rise time of a square wave signal applied toan electrode is sufficiently rapid to permit current measurement.

Wu et al. discloses using gated amperometric pulse sequences includingmultiple duty cycles of sequential excitation potentials and recoveries.See, US Patent Application Publication No. 2008/0173552. The excitationpotentials provide a constant voltage to an electrochemical cell. Acurrent response was generated during excitation potentials andmeasured. The current was reduced during recovery by at least half andpreferably to zero. The reduced recovery current was provided by an opencircuit condition to the electrochemical cell. Wu et al. thus disclosethat preferred recoveries are fundamentally different from applying azero potential recovery since these recoveries provide an independentdiffusion and analyte reaction during the recovery without the effectsof an applied electric potential even of zero volts.

A similar gated amperometric measurement method is disclosed by Wu,during which a recovery the electrical signal is in an off state thatincludes time periods when an electrical signal is not present but doesnot include time periods when an electrical signal is present butessentially has no amplitude. See, US Patent Application Publication No.2009/0145779. The off state was provided by opening an electricalcircuit mechanically, electrically, or by other methods. Moreover, USPatent Application Publication No. 2008/0179197 by Wu discloses gatedvoltammetric pulse sequences including multiple duty cycles ofsequential excitations and recoveries. The excitations provided alinear, cyclic or acyclic excitation to an electrochemical cell duringwhich response currents were measured while a applied potential wasvaried linearly with time. The recoveries also were provided in an opencircuit condition to the electrochemical cell.

Current methods and systems therefore provide some advantages withrespect to convenience; however, there remains a need for new methods ofelectrochemically measuring an analyte in a fluid sample even in thepresence of confounding variables.

BRIEF SUMMARY

In view of the disadvantages noted above, the disclosure describesmethods of electrochemically measuring an analyte in a fluidic samplesuch as a body fluid. The methods are based upon an inventive conceptthat includes using information derived from alternating current (AC)and/or DC responses, each designed to provide specific information aboutaspects of a biosensor and/or fluidic sample. For example, informationsuch as current response, shape and/or magnitude from an AC block oflow-amplitude signals can be used to correct for confounding variablessuch as Hct and/or temperature or to determine the condition of thebiosensor and its suitability for providing an accurate result.Alternatively, information such as a recovery current response, shapeand/or magnitude from a block of DC potentials can be used to correctfor not only Hct and/or temperature but also wetting of the reagent andsample diffusion. The inventive concept therefore provides certainadvantages, effects, features and objects when compared to known methodsof measuring an analyte concentration (or value) in a fluidic sample.

In one aspect, an electrochemical analysis method is provided formeasuring, determining, calculating or otherwise predicting an analyteconcentration in a fluidic sample that has been applied to anelectrochemical biosensor. The method can include at least a step ofproviding a test sequence of at least one DC block to the fluidicsample, where the test block is designed to elicit specific informationabout different aspects of the sample and/or the biosensor, where the DCblock includes at least one excitation potential and at least onerecovery potential, and where a closed circuit condition of an electrodesystem of the electrochemical biosensor is maintained during the DCblock.

In another aspect, an electrochemical analysis method is provided formeasuring, determining, calculating or otherwise predicting an analyteconcentration in a fluidic sample that has been applied to anelectrochemical biosensor. The method can include at least a step ofproviding a test sequence of at least one AC block and at least one DCblock to the fluidic sample, where each test block is designed to elicitspecific information about different aspects of the sample and/or thebiosensor.

With respect to the AC block, it can be a block of low-amplitude signalsapplied sequentially or simultaneously in parallel. In some instances,the AC block includes at least two different low-amplitude signals. Forexample, the AC block can include two (2) segments at two (2)frequencies such as, for example, about 10 kHz or about 20 kHz followedby about 1 kHz or about 2 kHz. In other instances, the AC block includesa plurality of low-amplitude signals. For example, the AC block can havefive (5) segments at four (4) frequencies such as, for example, about 10kHz, about 20 kHz, about 10 kHz, about 2 kHz and about 1 kHz.Alternatively, the AC block can have four (4) segments at four (4)frequencies such as, for example, about 20 kHz, about 10 kHz, about 2kHz and about 1 kHz. Alternatively, the AC block can have four (4)frequencies applied simultaneously at about 10 kHz, about 20 kHz, about10 kHz, about 2 kHz and about 1 kHz. Alternately still, the AC block canhave a multi-frequency excitation waveform that simultaneously appliesdesired low-amplitude AC signals.

In some instances, the AC block is applied for about 500 msec to about1.5 sec. In other instances, the AC block is applied for about 100 msecto about 300 msec.

In some instances, only one AC block is applied at a beginning of a testsequence. In other instances, however, additional AC blocks can beemployed and even can be interspersed with the at least one DC block. Assuch, the AC block can be applied before the DC block, after the DCblock, or interspersed within the DC block.

While the AC segments can be applied sequentially, they also can beco-added, and the combined frequencies can be applied simultaneously ina finite block where response information can be obtained by performinga Fourier transform to obtain phase and admittance amplitude informationfor each AC frequency.

Regardless of the segments, frequencies and their duration, AC currentresponse information can be obtained (i.e., measured) at any time duringthe AC block. In some instances, a series of AC current responsemeasurements can be performed early in the test sequence. Measurementstaken shortly after a sample is applied will be influenced by diffusion,temperature and reagent solubility. In other instances, AC measurementscan be obtained at a sufficient time after an adequate sample has beenapplied to allow the response to stabilize, and avoid the transientresponse in the first second. In yet other instances, the AC responsecurrent information can be obtained up to about 160,000/sec.

In some instances, the DC block is a continuous, unipolar excitationwaveform (i.e., the potential is applied and controlled throughout theDC block in a closed circuit), which is in contrast to some pulsedamperometric methods that employ an open circuit between excitationpulses. Likewise, a continuous current response can be collected,thereby allowing use of more sophisticated digital signal processingmethods, such as noise filtering and signal enhancement.

In some instances, the DC block includes a plurality of short-durationexcitation pulses and recovery pulses optimized for detecting an analytesuch as glucose, the optimization pertaining to pulse duration, rampedtransitions between the excitation pulse and recovery pulse, number ofcurrent responses measured during each pulse, and where in each pulsecurrent response measurements are taken.

With respect to the DC block, it can include at least one (1) pulse toabout ten (10) pulses at a potential that alternates between about 0 mVto about +450 mV. In some instances, the DC block can be a singlepotential step from about 0 mV to about +450 mV, where the potential ismaintained so that a decaying current response may be detected. That is,the DC block includes at least one excitation pulse and at least onerecovery pulse, where the pulses that alternate between about 0 mV toabout +450 mV.

Regardless of the number of pulses, each DC pulse can be applied forabout 50 msec to about 500 msec. Alternatively, each DC pulse at about+450 mV can be applied for about 250 msec, and each DC pulse at about 0mV can be applied for about 500 msec.

In some instances, the excitation pulses and the recovery pulses arecontrolled during a transition up/down using ramping between thepotentials. The pulses are controlled at a predetermined rate effectiveto mitigate a capacitive current response. Generally, the ramp rate isselected to provide about 50% or greater reduction in peak currentrelative to the peak current provided by a nearly ideal potentialtransition. For example, effective ramp rates can be from about 10mV/msec to about 50 mV/msec.

Regardless of the number of pulses, potentials and their duration, DCcurrent response information can be obtained at any time during the oneor more DC blocks. Moreover, the DC response current information can beobtained up to about 160,000/sec.

In some instances, only one DC block is used. In other instances, aplurality of DC blocks are used. For example, a first DC block can beused to detect an analyte of interest such as glucose, and a second DCblock can be used to provide information about a confounding variable.In some instances, the first DC block and the second DC block haveidentical ramp rates. In other instances, the second DC block has adistinct ramp rate when compared to the first DC block. Moreover, the DCblock can be a waveform using at least two different ramp rates.

In some instances, the second DC block can be used to detect the analyteand thus confirm the result from the first DC block. In other instances,other DC blocks can be used to measure other electro-active analytessuch as ketones in the sample.

Alternatively, the DC block can be a slow-ramp bi-polar (SRBP) waveformexcitation. In some instances, the waveform can be a triangularexcitation, whereas in another example it can be a sinusoidal ortrapezoidal excitation.

Alternatively still, the DC block can be a waveform having multiplefrequencies, and may be described by one of skill in the art ofelectrical engineering as a large-amplitude AC waveform, in contrast tothe low-amplitude AC block as described above.

In view of the foregoing, devices, apparatuses and systems used inconnection with electrochemical analysis are provided that incorporateone or more of the measurement methods disclosed herein. These devices,apparatuses and systems can be used to determine concentration ofanalytes including, but not limited to, amino acids, antibodies,bacteria, carbohydrates, drugs, lipids, markers, nucleic acids,peptides, proteins, toxins, viruses and other analytes, as well ascombinations thereof. In certain instances, the analyte is glucose.

These and other advantages, effects, features and objects of theinventive concept will become better understood from the descriptionthat follows. In the description, reference is made to the accompanyingdrawings, which form a part hereof and in which there is shown by way ofillustration, not limitation, embodiments of the inventive concept.

BRIEF DESCRIPTION OF THE DRAWINGS

The advantages, effects, features and objects other than those set forthabove will become more readily apparent when consideration is given tothe detailed description below. Such detailed description makesreference to the following drawings, wherein:

FIG. 1 shows an exemplary analyte measurement system including a meterand a biosensor.

FIG. 2 shows a simplified circuit diagram for an exemplary analytemeasurement system.

FIG. 3 shows an exemplary test sequence that may be employed by ananalyte measuring device, apparatus or system.

FIG. 4 is a graph of an exemplary test sequence of an analytemeasurement system.

FIG. 5 is a graph of an exemplary response of an analyte measurementsystem.

FIG. 6 is an enlarged view illustrating portions of the test sequence ofFIG. 4 and the response of FIG. 5.

FIG. 7A shows another exemplary test sequence that may be employed by ananalyte measurement system. FIG. 7B shows a more detailed exemplary testsequence that may be employed by the analyte measurement system.

FIG. 8 is a graph illustrating current responses for test samples withvarying Hct concentrations, constant temperatures, and constant glucoseconcentrations.

FIG. 9 is a graph illustrating current responses for test samples withvarying temperatures, constant Hct concentrations and constant glucoseconcentrations.

FIG. 10 is a graph illustrating current responses and current responsedeltas for a first exemplary test sequence.

FIG. 11 is a graph illustrating current responses and current responsedeltas for a second exemplary test sequence.

FIG. 12 is a graph illustrating another exemplary test signal andcurrent response deltas for the third exemplary test signal.

FIG. 13 is a graph illustrating another exemplary test signal.

FIG. 14 is a graph illustrating current responses and current responsedeltas to the fourth exemplary test signal of FIG. 13.

While the inventive concept is susceptible to various modifications andalternative forms, exemplary embodiments thereof are shown by way ofexample in the drawings and are herein described in detail. It should beunderstood, however, that the description of exemplary embodiments thatfollows is not intended to limit the inventive concept to the particularforms disclosed, but on the contrary, the intention is to cover alladvantages, effects, features and objects falling within the spirit andscope thereof as defined by the embodiments described herein and theclaims below. Reference should therefore be made to the embodimentsdescribed herein and claims below for interpreting the scope of theinventive concept. As such, it should be noted that the embodimentsdescribed herein may have advantages, effects, features and objectsuseful in solving other problems.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

The methods, devices, apparatuses and systems now will be described morefully hereinafter with reference to the accompanying drawings, in whichsome, but not all embodiments of the inventive concept are shown.Indeed, the inventive concept may be embodied in many different formsand should not be construed as limited to the embodiments set forthherein; rather, these embodiments are provided so that this disclosurewill satisfy applicable legal requirements.

Likewise, many modifications and other embodiments of the methods,devices, apparatuses and systems described herein will come to mind toone of skill in the art to which the disclosure pertains having thebenefit of the teachings presented in the foregoing descriptions and theassociated drawings. Therefore, it is to be understood that theinventive concept is not to be limited to the specific embodimentsdisclosed and that modifications and other embodiments are intended tobe included within the scope of the appended claims. Although specificterms are employed herein, they are used in a generic and descriptivesense only and not for purposes of limitation.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of skill in the artto which the disclosure pertains. Although any methods and materialssimilar to or equivalent to those described herein can be used in thepractice or testing of the present methods, devices, apparatuses andsystems, the preferred methods and materials are described herein.

Moreover, reference to an element by the indefinite article “a” or “an”does not exclude the possibility that more than one element is present,unless the context clearly requires that there be one and only oneelement. The indefinite article “a” or “an” thus usually means “at leastone.”

Overview

Analyte measuring methods are disclosed herein that use informationderived from AC and DC current responses to provide an analyteconcentration in a reliable manner. These measuring methods also can beused to reduce the effects of confounding variables such as Hct,temperature and/or variations in reagent thickness, thereby providing amore “true” analyte concentration.

The measurement methods disclosed herein largely utilize amperometry;however, it is contemplated that the methods can be used with otherelectrochemical measurement techniques (e.g., coulometry, potentiometryor voltammetry). Additional details regarding exemplary electrochemicalmeasurement methods are disclosed in, for example, U.S. Pat. Nos.4,008,448; 4,225,410; 4,233,029; 4,323,536; 4,891,319; 4,919,770;4,963,814; 4,999,582; 4,999,632; 5,053,199; 5,108,564; 5,120,420;5,122,244; 5,128,015; 5,243,516; 5,288,636; 5,352,351; 5,366,609;5,385,846; 5,405,511; 5,413,690; 5,437,999; 5,438,271; 5,508,171;5,526,111; 5,627,075; 5,628,890; 5,682,884; 5,727,548; 5,762,770;5,858,691; 5,997,817; 6,004,441; 6,054,039; 6,254,736; 6,270,637;6,645,368; 6,662,439; 7,073,246; 7,018,843; 7,018,848; 7,045,054;7,115,362; 7,276,146; 7,276,147; 7,335,286; 7,338,639; 7,386,937;7,390,667; 7,407,811; 7,429,865; 7,452,457; 7,488,601; 7,494,816;7,545,148; 7,556,723; 7,569,126; 7,597,793; 7,638,033; 7,731,835;7,751,864; 7,977,112; 7,981,363; 8,148,164; 8,298,828; 8,329,026;8,377,707; and 8,420,404, as well as RE36268, RE42560, RE42924 andRE42953.

Advantageously, the methods described herein can be incorporated intoSMBG devices, apparatuses and systems to more accurately and quicklyreport an analyte concentration, such as a glucose concentration,especially a blood glucose concentration.

Moreover, the measurement methods can be implemented using advancedmicroprocessor-based algorithms and processes that result indramatically improved system performance. These measurement methods alsooffer flexibility and number of ways to create algorithms that canachieve improved performance such as 10/10 performance. As used herein,“10/10 performance” means that a measured bG value is within about ±10%of the actual bG value for bG concentrations>100 mg/dL, and within ±10mg/dL of the actual bG value for bG concentrations<100 mg/dL.

Details regarding additional electrochemical measurement methods thatmay be useful in performing the methods disclosed herein can be found inthe following co-filed and co-pending patent applications titled:“METHODS OF SCALING DATA USED TO CONSTRUCT BIOSENSOR ALGORITHMS AS WELLAS DEVICES, APPARATUSES AND SYSTEMS INCORPORATING THE SAME” ApplicantDocket No. 31518; “METHODS OF FAILSAFING ELECTROCHEMICAL MEASUREMENTS OFAN ANALYTE AS WELL AS DEVICES, APPARATUSES AND SYSTEMS INCORPORATING THESAME” Docket No. 31520; “METHODS OF USING INFORMATION FROM RECOVERYPULSES IN ELECTROCHEMICAL ANALYTE MEASUREMENTS AS WELL AS DEVICES,APPARATUSES AND SYSTEMS INCORPORATING THE SAME” Docket No. 31522;“DESCRIPTOR-BASED METHODS OF ELECTROCHEMICALLY MEASURING AN ANALYTE ASWELL AS DEVICES, APPARATUSES AND SYSTEMS INCOPORATING THE SAME” DocketNo. 31523; and “METHODS OF DETECTING HIGH ANTIOXIDANT LEVELS DURINGELECTROCHEMICAL MEASUREMENTS AND FAILSAFING AN ANALYTE CONCENTRATIONTHEREFROM AS WELL AS DEVICES, APPARATUSES AND SYSTEMS INCORPORTING THESAME” Docket No. 31524.

Analyte Measurement Devices, Apparatuses and Systems

Prior to, and in connection with, describing the inventive measurementmethods, FIG. 1 shows an exemplary analyte measurement system includinga device such as a test meter 11 operatively coupled with anelectrochemical biosensor 20 (also known as a test element). Meter 11and biosensor 20 are operable to determine concentration of one or moreanalytes in a fluidic sample provided to the biosensor 20. In someinstances, the sample may be a body fluid sample such as, for example,whole blood, plasma, serum, urine or saliva. In other instances, thefluidic sample may be another type of sample to be tested for thepresence or concentration of one or more electrochemically reactiveanalyte(s) such as an aqueous environmental sample.

In FIG. 1, the biosensor 20 is a single use test strip removablyinserted into a connection terminal 14 of meter 11. In some instances,biosensor 20 is configured as a blood glucose test element and includesfeatures and functionalities for electrochemically measuring glucose. Inother instances, biosensor 20 is configured to electrochemically measureone or more other analytes such as, for example, amino acids,antibodies, bacteria, carbohydrates, drugs, lipids, markers, nucleicacids, peptides, proteins, toxins, viruses, and other analytes.

Meter 11 includes an electronic display 16 that is used to displayvarious types of information to the user including analyteconcentration(s) or other test results, and user interface 50 forreceiving user input. Meter 11 further includes a microcontroller andassociated test signal generating and measuring circuitry (not shown)that are operable to generate a test signal, to apply the signal to thebiosensor 20, and to measure one or more responses of the biosensor 20to the test signal. In some instances, meter 11 can be configured as ablood glucose measurement meter and includes features andfunctionalities of the ACCU-CHEK® AVIVA® meter as described in thebooklet “Accu-Chek® Aviva Blood Glucose Meter Owner's Booklet” (2007),portions of which are disclosed in U.S. Pat. No. 6,645,368. In otherinstances, meter 11 can be configured to electrochemically measure oneor more other analytes such as, for example, amino acids, antibodies,bacteria, carbohydrates, drugs, lipids, markers, nucleic acids,proteins, peptides, toxins, viruses, and other analytes. Additionaldetails regarding exemplary meters configured for use withelectrochemical measurement methods are disclosed in, for example, U.S.Pat. Nos. 4,720,372; 4,963,814; 4,999,582; 4,999,632; 5,243,516;5,282,950; 5,366,609; 5,371,687; 5,379,214; 5,405,511; 5,438,271;5,594,906; 6,134,504; 6,144,922; 6,413,213; 6,425,863; 6,635,167;6,645,368; 6,787,109; 6,927,749; 6,945,955; 7,208,119; 7,291,107;7,347,973; 7,569,126; 7,601,299; 7,638,095 and 8,431,408.

One of skill in the art understands that the measurement methodsdescribed herein can be used in other measurement, devices, apparatuses,systems and environments such as, for example, hospital test systems,laboratory test systems and others.

It shall be understood that the biosensor and meter can includeadditional and/or alternate attributes and features in addition to orinstead of those shown in FIG. 1. For example, the biosensor can be inthe form of a single use, disposable electrochemical test strip having asubstantially rectangular shape. It shall be appreciated that thebiosensors can include different forms such as, for example, test stripsof different configurations, dimensions or shapes, non-strip testelements, disposable test elements, reusable test elements,micro-arrays, lab-on-chip devices, bio-chips, bio-discs, bio-cds orother test elements. Additional details regarding exemplary biosensorsconfigured for use with electrochemical measurement methods aredisclosed in, for example, U.S. Pat. Nos. 5,694,932; 5,762,770;5,948,695; 5,975,153; 5,997,817; 6,001,239; 6,025,203; 6,162,639;6,245,215; 6,271,045; 6,319,719; 6,406,672; 6,413,395; 6,428,664;6,447,657; 6,451,264; 6,455,324; 6,488,828; 6,506,575; 6,540,890;6,562,210; 6,582,573; 6,592,815; 6,627,057; 6,638,772; 6,755,949;6,767,440; 6,780,296; 6,780,651; 6,814,843; 6,814,844; 6,858,433;6,866,758; 7,008,799; 7,063,774; 7,238,534; 7,473,398; 7,476,827;7,479,211; 7,510,643; 7,727,467; 7,780,827; 7,820,451; 7,867,369;7,892,849; 8,180,423; 8,298,401; 8,329,026, as well as RE42560, RE42924and RE42953.

FIG. 2 shows a simplified circuit diagram 400 of an exemplary analytemeasurement system including a biosensor 420 operatively coupled with ameter 410 to provide electrical communication between biosensor 420 andmeter 410. Biosensor 420 includes a test cell 421 having a workingelectrode 422 and a counter electrode 423 in contact with a combinedreagent and sample 424. Working electrode 422 is in electricalcommunication with the negative input of amplifier 414 of meter 410.Counter electrode 423 is in electrical communication with a virtualground or reference potential of meter 410.

Meter 410 includes a microcontroller 411, which is operable to generateand output a test control signal at output 412. The test control signaldrives amplifier 413 to output a test potential to the positive input ofamplifier 414. This test potential is also seen at the negative input ofamplifier 414 due to a virtual short between the positive input andnegative input of amplifier 414. The test potential present at thenegative input of amplifier 414 provided to working electrode 422. Thus,the test control signal output by microcontroller 411 is operable tocontrol the test potential applied to the working electrode 422. Thetest control signal provided at output 412 and test potential providedto working electrode 422 may include a number features such as ACcomponents, preconditioning components, and DC pulse sequences includingexcitation potentials and closed circuit recovery potentials, examplesof which are further described herein below.

The test potential applied to working electrode 422 produces a currentresponse 450 that is provided to the negative input of amplifier 414.Amplifier 414 is configured as an I/V converter and outputs a voltage toinput 460 of microcontroller 411 that is proportional to currentresponse 450. Microcontroller 411 detects the voltage at input 460 anddetermines the current response 450 by dividing the voltage seen atinput 460 by the value of gain resistor 415. The current response 450may include responses to test potentials including AC components,preconditioning components, and DC pulse sequences including excitationpotentials and closed circuit recovery potentials, examples of which arefurther described herein below.

It shall be appreciated that additional exemplary analyte measurementsystems may include a number of features in addition to or asalternatives to those illustrated in simplified circuit diagram 400. Forexample, microcontroller 411 also may be operatively connected to othercomponents of meter 410 such as one or more digital memories, displays,and/or user interfaces, such as those illustrated and described above inconnection with FIG. 1, as well as controller and driver circuitryassociated therewith. In FIG. 2, output 412 is an analog outputconnected to a D/A converter internal to microcontroller 412, and input460 is an analog input connected to an A/D converter internal tomicrocontroller 412. In other instances, output 412 may be a digitaloutput connected to an external D/A converter and input 460 may be adigital input connected to an external A/D converter. In FIG. 2, testcell 421 is a two-electrode test cell; however, other test cells can bethree-electrode test cells, or other electrode systems.

In FIG. 2, a test potential can be applied to a working electrode toprovide a potential difference between the working electrode and acounter electrode. Alternatively, a test potential other than virtualground or reference potential can be provided as a counter electrode toprovide a potential difference between the working electrode and acounter electrode. It shall be appreciated that the foregoing and avariety of other additional and alternate test cell, electrode, and/orcircuitry configurations operable to apply a test signal to an electrodesystem in contact with a combined sample and reagent and measure aresponse thereto may be utilized.

Measurement Methods

As noted above, the measurement methods described herein are based uponan inventive concept that includes using information derived from ACand/or DC current responses to a test sequence having at least one ACblock and/or at least one DC block, each block designed to providespecific information about aspects of a fluidic sample and/or biosensor.

The methods generally include applying to a fluidic sample, such as abody fluid, an AC block of low-amplitude signals in connection with a DCblock having a controlled, pulsed sequence and measuring the AC and DCcurrent responses. FIG. 3 shows an exemplary test sequence and responsethereto that may be used in connection with SMBGs and other testsystems. The test sequence thus includes an AC block of low-amplitudesignals followed by a controlled, DC block of excitation pulses andrecovery pulses.

With respect to the AC block, it can include a plurality of AC segmentssuch as, for example, from about 2 segments to about 10 segments, fromabout 3 segments to about 9 segments, from about 4 segments to about 8segments, from about 5 segments to about 7 segments, or about 6segments. In other instances, the AC block can include about 2 segments,about 3 segments, about 4 segments, about 5 segments, about 6 segments,about 7 segments, about 8 segments, about 9 segments, or about 10segments. In still other instances, the AC block can have more than 10segments, that is, about 15 segments, about 20 segments, or about 25segments. In yet other instances, the AC block can include 1 segment,where the segment has multiple low-amplitude AC signals appliedsimultaneously.

One of skill in the art understands that the number of segments in theAC block will be limited by the complexity of the response, theassociated frequency range and time available to perform themeasurements. Higher frequencies generally require high bandwidthelectronics and faster sampling, whereas lower frequencies take longerand are typically noisier. The maximum number of segments therefore willbe a compromise of these parameters, choosing the minimum count andfrequency span needed to discriminate the sample and environmentaland/or confounding factors of interest.

As used herein, “about” means within a statistically meaningful range ofa value or values such as a stated concentration, length, molecularweight, pH, potential, time frame, temperature, voltage or volume. Sucha value or range can be within an order of magnitude, typically within20%, more typically within 10%, and even more typically within 5% of agiven value or range. The allowable variation encompassed by “about”will depend upon the particular system under study, and can be readilyappreciated by one of skill in the art.

The frequency of each signal in each segment of the AC block can be fromabout 1 kHz to about 20 kHz, from about 2 kHz to about 19 kHz, fromabout 3 kHz to about 18 kHz, from about 4 kHz to about 17 kHz, fromabout 5 kHz to about 16 kHz, from about 6 kHz to about 15 kHz, fromabout 7 kHz to about 14 kHz, from about 8 kHz to about 13 kHz, fromabout 9 kHz to about 12 kHz or from about 10 kHz to about 11 kHz. Inother instances, the frequency of each segment in the AC block can beabout 1 kHz, about 2 kHz, about 3 kHz, about 4 kHz, about 5 kHz, about 6kHz, about 7 kHz, about 8 kHz, about 9 kHz, about 10 kHz, about 11 kHz,about 12 kHz, about 13 kHz, about 14 kHz, about 15 kHz, about 16 kHz,about 17 kHz, about 18 kHz, about 19 kHz, or about 20 kHz. In stillother instances, the frequency of each signal in each segment of the ACblock can be more than 20 kHz, that is, about 30 kHz, about 40 kHz, orabout 50 kHz. In some instances, one or more of the segments can havethe same frequency, whereas in other instances each segment has adistinct frequency from the other segments. Four frequencies, however,generally is adequate. The exact frequencies employed can be readilygenerated by simple integer division of a measurement system clock'smaximum frequency.

A maximum frequency limit for a signal in a segment of the AC block,however, can be up to about 100 kHz for an inexpensive, battery-poweredhandheld instrument. Beyond that, the increasing demands on analogbandwidth, sampling rate, storage and processing speed quickly add up,while the imaginary portion of a typical biosensor response becomesincreasingly smaller with frequency. Lower frequencies have longerperiods and take longer times to sample with comparable accuracy.

The AC block typically includes at least two different low-amplitudesignals. For example, the AC block can include two (2) segments at two(2) frequencies such as, for example, about 10 kHz or about 20 kHzfollowed by about 1 kHz or about 2 kHz. In other instances, the AC blockincludes a plurality of low-amplitude signals. For example, the AC blockcan have five (5) segments at four (4) frequencies such as, for example,about 10 kHz, about 20 kHz, about 10 kHz, about 2 kHz and about 1 kHz.Alternatively, the AC block can have four (4) segments at four (4)frequencies such as, for example, about 20 kHz, about 10 kHz, about 2kHz and about 1 kHz. Alternatively, the AC block can have four (4)frequencies applied simultaneously at about 10 kHz, about 20 kHz, about10 kHz, about 2 kHz and about 1 kHz. Alternately still, the AC block canhave a multi-frequency excitation waveform that simultaneously appliesthe desired low-amplitude AC signals. The AC frequencies may be appliedsequentially, or combined and applied simultaneously and analyzed viaFourier Transform.

The AC block can be applied for about 500 msec to about 1.5 sec, about600 msec to about 1.25 sec, about 700 msec to about 1000 msec, or about800 msec to about 900 msec. Alternatively, the AC block can be appliedfor about 500 msec, about 600 msec, about 700 msec, about 800 msec,about 900 msec, about 1000 msec, about 1.25 sec or about 1.5 sec. Inparticular, the AC block can be applied for about 100 msec to about 300msec.

One of skill in the art, however, understands that the number,frequency, duration and order of the AC segments can be varied.

AC current response information can be obtained at any time during atest sequence. Impedance results at lower frequencies may be influencedby analyte concentration if obtained after an electrochemical cell is DCpolarized. In some instances, a series of AC current responsemeasurements can be obtained early in the test sequence. Measurementstaken shortly after a fluidic sample is applied to a biosensor will beinfluenced by diffusion, temperature and reagent solubility. In otherinstances, the AC response current measurements can be obtained at asufficient time after an adequate sample has been applied to allow theresponse to stabilize, and avoid the transient response in the firstsecond. Likewise, response current measurements can be made at one ormore frequencies. Due to their capacitive nature, multiple ACmeasurements separated by a frequency octave or decade may offerdifferent sensitivities or easier manipulation.

Accurate measurements can be more difficult at frequencies above 5 kHzbut can offer increased visibility to the real impedance. Here,frequencies at 20 kHz, 10 kHz, 2 kHz and 1 kHz were used. In addition,the frequencies were applied in this sequence to simplify measurementelectronics and to allow the lower frequencies (larger imaginaryimpedances) longer to stabilize.

Additional details regarding exemplary AC blocks in electrochemicalmeasurement methods are disclosed in, for example, U.S. Pat. Nos.7,338,639; 7,390,667; 7,407,811; 7,417,811; 7,452,457; 7,488,601;7,494,816; 7,597,793; 7,638,033; 7,751,864; 7,977,112; 7,981,363;8,148,164; 8,298,828; 8,377,707 and 8,420,404.

With respect to the DC block, it typically includes a constantly appliedpotential difference that alternates between about 0 mV and apredetermined positive potential difference, or other slowlytime-varying potential difference that can be analyzed by traditional DCelectrochemical methods. One of skill in the art, however, understandsthat the range for the applied potential difference can, and will, varydepending upon the analyte and reagent chemistry used.

The DC block can include a plurality of pulses such as, for example,from about 2 pulses to about 10 pulses, from about 3 pulses to about 9pulses, from about 4 pulses to about 8 pulses, from about 5 pulses toabout 7 pulses, or about 6 pulses. In other instances, the DC block caninclude about 2 pulses, about 3 pulses, about 4 pulses, about 5 pulses,about 6 pulses, about 7 pulses, about 8 pulses, about 9 pulses, or about10 pulses. In still other instances, the DC block can have more than 10pulses, that is, about 15 pulses, about 20 pulses, or about 25 pulses.As used herein, “pulse” means at least one excitation and/or onerecovery period. The number of pulses, however, typically is limited bythe available time for the test sequence. Shorter durations probefurther from the electrode surface, and increase sensitivity to reagentthickness and diffusion modifiers.

The potential of each pulse in the DC block can be from about 0 mV toabout 450 mV, from about 10 mV to about 425 mV, from about 15 mV toabout 400 mV, from about 20 mV to about 375 mV, from about 25 mV toabout 350 mV, from about 30 mV to about 325 mV, from about 35 mV toabout 300 mV, from about 40 mV to about 275 mV, from about 45 mV toabout 250 mV, from about 50 mV to about 225 mV, from about 75 mV toabout 200 mV, from about 100 mV to about 175 mV, or from about 125 mV toabout 150 mV. In other instances, the potential of each pulse in the DCblock can be about 1 mV, about 10 mV, about 15 mV, about 20 mV, about 25mV, about 30 mV, about 35 mV, about 40 mV, about 45 mV, about 50 mV,about 60 mV, about 70 mV, about 80 mV, about 90 mV, about 100 mV, about110 mV, about 120 mV, about 130 mV, about 140 mV, about 150 mV, about160 mV, about 170 mV, about 180 mV, about 190 mV, about 200 mV, about210 mV, about 220 mV, about 230 mV, about 240 mV, about 250 mV, about260 mV, about 270 mV, about 280 mV, about 290 mV, about 300 mV, about310 mV, about 320 mV, about 330 mV, about 340 mV, about 350 mV, about360 mV, about 370 mV, about 380 mV, about 390 mV, about 400 mV, about410 mV, about 420 mV, about 430 mV, about 440 mV, or about 450 mV. Instill other instances, the potential of each pulse of the DC block canbe more than 450 mV, that is, about 475 mV, about 500 mV, about 525 mV,about 550 mV, about 575 mV, about 600 mV kHz, about 625 mV, about 650mV, about 675 mV, about 700 mV, about 725 mV, or about 750 mV. In stillother instances, the excitation pulse potential can be greater-than,less-than or equal to about +450 mV. In some instances, one or more ofthe pulses can have the same potential, whereas in other instances eachpulse has a distinct potential from the other pulses.

As noted above, the applied DC potential can be fixed at about 0 mVbetween excitation pulses to provide a recovery pulse, thus making it agenerally continuous, unipolar excitation waveform. This is in contrastto a test signal sequence from known techniques that prescribe the useof an open circuit between positive DC pulses, thereby excluding thepossibility of collecting and analyzing the current between positivepulses.

Regardless of the number, each DC pulse can be applied for about 50 msecto about 500 msec, about 60 msec to about 450 msec, about 70 msec toabout 400 msec, about 80 msec to about 350 msec, about 90 msec to about300 msec, about 100 msec to about 250 msec, about 150 msec to about 200msec, or about 175 msec. Alternatively, each pulse can be applied forabout 50 msec, about 60 msec, about 70 msec, about 80 msec, about 90msec, about 100 msec, about 125 msec, about 150 msec, about 175 msec,about 200 msec, about 225 msec, about 250 msec, about 275 msec, about300 msec, about 325 msec, about 350 msec, about 375 msec, about 400msec, about 425 msec, about 450 msec, about 475 msec or about 500 msec.In particular, each DC pulse at +450 mV can be applied for about 250msec, and each DC pulse at 0 mV can be applied for about 500 msec.Alternatively still, each pulse can be applied for less than about 50msec or more than about 500 msec. The duration should be long enough orthe onset soft enough to avoid charging currents. Regardless, the pulseduration should be applied long enough to enable reasonable 50/60 Hznoise rejection. Moreover, the time between pulses is ideally longenough to allow the electrochemical cell to discharge and return closeto its pre-pulse state. Furthermore, the operating potential will dependupon the mediator and measurement system. The examples hereindemonstrate proof-of-principal with NA-derived redox mediator.

Generally, the ramp rate of each pulse is selected to provide about 50%or greater reduction in peak current relative to the peak currentprovided by a nearly ideal potential transition. In some instances, eachpulse can have the same ramp rate. In other instances, some pulses canhave the same ramp rate and other pulses can have a different ramp rate.In still other instances, each pulse has its own ramp rate. For example,effective ramp rates can be from about 5 mV/msec to about 75 mV/msec orfrom about 10 mV/msec to about 50 mV/msec, 15 mV/msec to about 25mV/msec, or about 20 mV/msec. Alternatively, the ramp rate can be about5 mV/msec, about 10 mV/msec, about 15 mV/msec, about 20 mV/msec, about25 mV/msec, about 30 mV/msec, about 35 mV/msec, about 40 mV/msec, about45 mV/msec, about 50 mV/msec, about 55 mV/msec, about 60 mV/msec, about65 mV/msec, about 70 mV/msec, or about 75 mV/msec. In particular, theramp rate can be from about 40 mV/msec to about 50 mV/msec.

To determine the excitation potential for a given redox mediator, onemay plot current measured a fixed time after a selected workingelectrode/counter-electrode (WE-CE) potential step is applied (e.g., 3.5sec). In any case, one of skill in the art would strive to operatecomfortably on a current-potential plateau. Higher potentials, however,are not always better as they can invite other (i.e., interfering)reactions that may undesirably contribute to the analyte measurement ofinterest.

In some instances, the test sequence includes a single DC block, whereasin other instances the test sequence includes two or more DC blocks.

An exemplary DC block can alternate (i.e., pulse) between about 0 mV andabout +450 mV (in biamperometric mode).

Like the AC block, one of skill in the art understands that the number,potential, duration and order of the DC pulses can be varied.

In the methods, the AC and/or DC response current information can beobtained (i.e., measured or recorded) at about 2,000/sec to about200,000/sec, at about 3,000/sec to about 190,000/sec, at about 4,000/secto about 180,000/sec, at about 5,000/sec to about 170,000, at about6,000/sec to about 160,000/sec, at about 7,000/sec to about 150,000/sec,at about 8,000/sec to about 140,000/sec, at about 9,000/sec to about130,000/sec, at about 10,000/sec to about 120,000/sec, at about15,000/sec to about 110,000/sec, at about 20,000/sec to about100,000/sec, at about 30,000/sec to about 90,000/sec, at about40,000/sec to about 80,000/sec, at about 50,000/sec to about 70,000/sec,or at about 60,000/sec. In some instances, the AC and/or DC responsecurrent information can be obtained at about 100/sec to about 200/sec,at about 200/sec to about 300/sec, at about 300/sec to about 400/sec, atabout 400/sec to about 500/sec, at about 500/sec to about 600/sec, atabout 600/sec to about 700/sec, at about 700/sec to about 800/sec, atabout 800/sec to about 900/sec, at about 1,000/sec to about 1,500/sec,at about 1,500/sec to about 2,000/sec, at about 2,000/sec to about2,500/sec, at about 2,500/sec to about 3,000/sec, at about 3,000/sec toabout 3,500/sec, at about 3,500/sec to about 4,000/sec, at about4,000/sec to about 4,500/sec, at about 4,500/sec to about 5,000/sec, atabout 5,000/sec to about 5,500/sec, at about 5,500/sec to about6,000/sec, at about 6,000/sec to about 6,500/sec, at about 6,500 toabout 7,000/sec, at about 7,000/sec to about 7,500/sec, at about7,500/sec to about 8,000/sec, at about 8,000/sec to about 8,500/sec, atabout 8,500 to about 9,000/sec, at about 9,000/sec to about 9,500/sec,at about 9,500/sec to about 10,000/sec, at about 10,000/sec to about20,000/sec, at about 20,000/sec to about 30,000/sec, at about 30,000/secto about 40,000/sec, at about 40,000/sec to about 50,000/sec, at about50,000/sec to about 60,000/sec, at about 60,000/sec to about 70,000/sec,at about 70,000/sec to about 80,000/sec, at about 80,000/sec to about90,000/sec, at about 90,000/sec to about 100,000/sec, at about100,000/sec to about 110,000/sec, at about 110,000/sec to about120,000/sec, at about 120,000/sec to about 130,000/sec, at about130,000/sec to about 140,000/sec, at about 140,000/sec to about150,000/sec, at about 150,000/sec to about 160,000/sec, at about160,000/sec to about 170,000/sec, at about 170,000/sec to about180,000/sec, at about 180,000/sec to about 190,000/sec, or at about200,000/sec. In other instances, the AC and/or DC response currentinformation can be obtained up to about 100/sec, about 200/sec, about300/sec, about 400/sec, about 500/sec, 600/sec, about 700/sec, about800/sec, about 900/sec, about 1,000/sec, about 1,250/sec, about1,500/sec, about 1,750/sec, about 2,000/sec, about 2,225/sec, about2,500/sec, about 2,750/sec, about 3,000/sec, about 3,250/sec, about3,500/sec, about 3,750/sec, about 4,000/sec, about 4,250/sec, about4,500/sec, about 4,750/sec, about 5,000/sec, about 5,250/sec, about5,500/sec, about 5,750/sec, about 6,000/sec, about 6,250/sec, about6,500, about 7,000/sec, about 7,250/sec, about 7,500/sec, about7,750/sec, about 8,000/sec, about 8,250/sec, about 8,500/sec, about8,750, about 9,000/sec, about 9,250/sec, about 9,500/sec, about9,750/sec, about 10,000/sec, about 15,000/sec, about 20,000/sec, about25,000/sec, about 30,000/sec, about 35,000/sec, about 40,000/sec, about45,000/sec, about 50,000/sec, about 55,000/sec, about 60,000/sec, about65,000/sec, about 70,000/sec, about 75,000/sec, about 80,000/sec, about85,000/sec, about 90,000/sec, about 95,000/sec, about 100,000/sec, about105,000/sec, about 110,000/sec, about 115,000/sec, about 120,000/sec,about 125,000/sec, about 130,000/sec, about 135,000/sec, about140,000/sec, about 145,000/sec, about 150,000/sec, about 155,000/sec,about 160,000/sec, about 165,000/sec, about 170,000/sec, about175,000/sec, about 180,000/sec, about 185,000/sec, about 190,000/sec,about 195,000 or at about 200,000/sec. In yet other instances, the ACand/or DC response current information can be obtained at more than200,000/sec.

Taken together, the at least one AC block and the at least one DC blockencompass the test sequence that is applied to the sample. Asillustrated in FIG. 3, an exemplary test sequence therefore can include:(1) an AC block of a plurality of AC segments at different frequencies;and (2) a DC block of short-duration (e.g., about 50-500 msec) about450-mV pulses separated by similarly short-duration (e.g., about 50-500msec) recovery pulses, during which a closed circuit about 0-mV recoverypotential is applied.

Stated differently, an exemplary test sequence therefore can includethree (3) blocks that may be executed in any order. A first AC block canbegin after a fluidic sample is detected and confirmed on a biosensor.The initial first sec is a 10 kHz, small amplitude AC waveform tomonitor the dynamics of reagent dissolution and reaction dynamics. Thisis followed by four (4) 150 msec AC segments of 20 kHz, 10 kHz, 2 kHzand 1 kHz. A second block can be a series of DC pulses, where a positiveapplied potential difference is of sufficient amplitude and duration toinduce a diffusion-limited current. These pulses are interlaced with alesser potential difference that are not of a sufficient amplitude toinduce a diffusion-limited current and long enough to allow the cell toreturn near its pre-pulse state. A third block also can be a series ofDC pulses, such as an SRBP.

Current response information is collected from the test sequence andincludes current responses to the AC and DC blocks. In some instances,the current response information can be collected at an A/D samplingrate for DC and AC measurements to simplify the system design, includinga single shared signal path for AC and DC measurements. Common digitalaudio sampling rates range include, but are not limited to, from about44.1 kHz to about 192 kHz. A/D converters in this range are readilyavailable from variety of commercial semiconductor suppliers.

Current response information to the AC block can be used to determineimpedance, admittance and phase values or other complex parameters asdescribed in further detail below. Likewise, current information to theDC block can be used to determine analyte concentration or other complexparameters as described in further detail below.

As shown in FIGS. 4-5, one trace illustrates the applied DC potential,and the other trace illustrates the AC and DC current responses,respectively. As noted above, the applied DC potential can be fixed atabout 0 mV between pulses to provide a recovery pulse, thus making it agenerally continuous excitation waveform. This is in contrast to a testsequence from known techniques that prescribe the use of an open circuitbetween positive DC pulses, thereby excluding the possibility ofcollecting and analyzing the current between positive excitation pulses.As used herein, “recovery pulse” means an about zero-potential pulseapplied for an adequately long recovery period in which theelectrochemical reaction with the analyte of interested (e.g., glucose)is turned “off,” thereby allowing the system to return to a fixedstarting point before subsequent interrogation with another morepositive DC pulse.

As part of the inventive concept, it has been recognized that therecovery responses in the DC block include unique informational content,particularly pertaining to Hct and temperature. Furthermore, thisinformation provides value and can be utilized to further refineaccuracy and performance of SMBG devices, apparatuses and systems.

Returning to FIG. 4, the responses to the pulsed DC block encode Hct andtemperature information, as well as real-time information about otherimportant processes, such as wetting of the reagent, sample diffusionand separation with respect to the reagent, the establishment of astable glucose transport gradient, and the kinetics associated with thereducible analyte. The illustrated DC block provides short, distinctstrobing of these processes with respect to time. Each positive DC pulseproduces a distinct current signature, which is not exactly like theothers due to its position in time.

Importantly, each closed circuit recovery potential pulse provides anadequately long recovery period in which the electrochemical reactionwith glucose is turned off, thereby allowing the system to return to acommon starting point before subsequent interrogation with anotherpositive pulse.

Just as the shapes of the current decays from positive DC pulses encodeinformation about glucose, Hct and temperature (as well as the otherbiosensor processes noted above), the shapes of the recovery pulses alsoare unique. Each recovery pulse produces a negative current responsewith a rate of growth that also encodes distinct, time-orderedinformation describing how the biamperometric system returns to a givenreference state. The rate of current growth during the recovery pulse isnot simply a mirror image of the current decay associated with aneighboring positive DC pulse, because the glucose reaction has beenturned off by selecting a potential magnitude that cannot initiate andsustain the electrochemical reaction with glucose. The exemplarymeasurement methods disclosed herein use unique information contentpertaining to Hct, temperature and other confounding variables encodedby the recovery current responses to improve the accuracy andperformance SMBG devices, apparatuses and systems.

It shall be appreciated that near-zero, and non-zero positive andnegative potential magnitudes also may be used as recovery pulses inadditional embodiments, and that the magnitude, duration, and shapes ofall pulses may vary from the illustrated exemplary embodiments. It shallalso be appreciated that the exemplary embodiments disclosed herein donot restrict the number of AC signals that may be employed, theirpositions in time, or their amplitude(s)/frequencies. Nor does itrestrict interspersing AC signals within the pulsed DC block of the testsequence, such as in the exemplary test signal illustrated in FIG. 4 anddiscussed in greater detail below. Furthermore, the exemplaryembodiments disclosed herein do not restrict the number, length ormagnitude of the DC pulses.

FIG. 4 shows an exemplary test sequence 500 that can be provided to anelectrode system of an electrochemical test cell. The vertical axis 501of graph denotes working electrode potential in volts (V). It shall beunderstood that working electrode potential may refer to a potentialapplied to a working electrode or to a potential difference between aworking electrode and another electrode such as a counter or referenceelectrode regardless of the electrode or electrodes to which a potentialor a test signal is applied. The horizontal axis 502 of graph denotestime in sec. Test sequence 500 is applied at or after time=0 sec, whichis a time at which a sufficient sample is present in a test cell as maybe determined using sample sufficiency detection electrodes and signalsor through other methods.

Test sequence 500 begins with a signal component 510 (or block) that mayinclude one or more AC segment(s), preconditioning test segment(s) orcombinations thereof. Signal component 510 also may include incubationsignal components that are selected not to drive an electrochemicalreaction but to allow for reagent hydration and progression of reactionkinetics. Such incubation components may include, for example, an opencircuit condition, a 0 mV potential, a substantially 0 mV averagepotential, or a non-zero volt potential such as a non-zero potentialthat is less than the potential needed to drive a particular reaction ofinterest.

In some instances, signal component 510 comprises one or more ACsequences and frequencies provided to an electrode system of anelectrochemical test cell. For example, the AC segments of signalcomponent 510 include a 10 kHz segment applied from about time=0 sec toabout time=1.2 sec, a 20 kHz segment applied from about time=1.2 sec toabout time=1.3 sec, a 10 kHz segment applied from about time=1.3 sec toabout time=1.4 sec, a 2 kHz segment applied from about time=1.4 sec toabout time=1.5 sec, and a 1 kHz segment applied from about time=1.5 secto about time=1.6 sec. Alternatively, the AC segments and frequencies ofsignal component 510 includes a 10 kHz signal applied for about 1.5 sec,followed by a 20 kHz signal applied for about 0.2 sec, followed by a 10kHz signal applied for about 0.2 sec, followed by a 2 kHz signal appliedfor about 0.2 sec, followed by a 1 kHz signal applied for about 0.2 sec.

As noted above, the signal component 510 can include one or morepreconditioning signal(s). In some instances, the signal component 510includes a positive DC preconditioning pulse applied starting at abouttime=0 sec for about 200-600 msec and having an amplitude of about 100mV or greater. In other instances, the signal component 510 can includea positive DC preconditioning pulse applied starting at about time=0 secfor about 500 msec and having an amplitude of about 450 mV. In stillother instances, the signal component 510 can include a two-cycletriangular potential waveform including a ramp rate of about 2 mV/msec.

As such, the signal component 510 can include combinations of one ormore AC segments as well as preconditioning signal component(s). In someinstances, the signal component 510 includes one or more AC signalcomponents followed by one or more preconditioning signal components. Inother instances, the signal component 510 includes one or morepreconditioning signal components followed by one or more AC signalcomponents.

After signal component 510, a pulsed DC sequence 520 (or block) isapplied to the electrode system. Pulse sequence 520 begins with theworking electrode potential being ramped up to the excitation potentialof pulse 521. From pulse 521 the working electrode potential is rampeddown to the recovery potential of pulse 522. From potential 522 theworking electrode potential is sequentially ramped up and down to thepotentials of pulses 523-532. As shown in FIG. 4, the ramp rate betweenpulses is controlled to occur at a predetermined rate effective tomitigate capacitive current response. In some instances, the ramp rateis selected to provide a 50% or greater reduction in peak currentrelative to the peak current provided by a substantially square waveexcitation in which signal rise time is determined by the nativecharacteristics of the driving circuitry rather than being deliberatelycontrolled according to a predetermined target rate or range.

Pulses 521, 523, 525, 527, 529 and 531 are examples of ramp-ratecontrolled excitation potential pulses that provide an excitationpotential to an electrochemical test cell effective to drive anelectrochemical reaction in the test cell and generate an associatedFaradaic current response which may be convolved with capacitivecharging current responses and other current response informationattributable to a plurality of confounding variables. As also shown inFIG. 4, the excitation potential pulses provide a potential differencebetween a working electrode and a counter electrode of about 450 mV thatis about 130 msec in duration. The excitation potential shown isselected to drive a particular analyte reaction, which in this case isan enzyme-mediated reaction of glucose. It shall be understood that themagnitude and duration of the excitation potential pulses may varydepending upon the particular activation potential of the mediator usedor the potential needed to drive a particular reaction of interest.

Pulses 522, 524, 526, 528, 530 and 532 are examples of closed circuitrecovery potential pulses that provide a potential to a workingelectrode of an electrochemical test cell during which a closed circuitcondition of the test cell is maintained to control the test cell todischarge current and to more rapidly restore test cell conditions to asubstantially common starting point for subsequent interrogation with anexcitation potential pulse. Closed circuit recovery potential pulsesalso may be ramp rate controlled in the same or a similar manner toexcitation potential pulses. As shown in FIG. 4, the recovery potentialpulses provide a potential difference between a working electrode and acounter electrode of about 0 mV, which is about 280 msec in durationduring which the electrode system is maintained in a closed circuitcondition.

In some instances, the magnitude of the DC potential provided by aclosed circuit recovery pulse and its duration may vary depending uponthe potential below which a test cell can recover toward apre-excitation state and the time needed to provide a desired response.Thus, some embodiments can include recovery potential pulses having anon-zero potential that is less than the activation potential of a givenmediator. Some instances include recovery potential pulses having anon-zero potential that is less than the potential needed to drive aparticular reaction of interest. Other instances include recoverypotential pulses having a non-zero potential that is less than theminimum redox potential for a specified reagent system. Still otherinstances include recovery potential pulses having an average potentialof about 0 mV, but which have pulse portions greater than about 0 mV andportions less than about 0 mV. Still other instances include recoverypotential pulses having an average potential according to any of theaforementioned non-zero potentials, but which have portions greater thanthe non-zero average and portions less than the non-zero average.

FIG. 5 shows a response signal 600 produced by a test cell in responseto test signal 500 of FIG. 4. The vertical axis 601 of graph 600 denotesworking electrode current in μA. The horizontal axis 602 of graph 600denotes time in seconds. Current response 600 begins with responsecomponent 610 that includes a response to signal component 510. In someinstances, response component 610 includes AC current responses fromwhich impedance, admittance and phase angle can be determined. Suchmeasurements may be performed for one or more AC block segments orcomponents such as those described above in connection with FIG. 4. Insome instances, signal component 610 includes a preconditioning signalcomponent but no AC segment and no measurement of response component 610is performed. In other instances, signal component 610 includes acombination of the foregoing and/or other components.

After response component 610, response signal 600 includes a sequence ofexponentially decaying excitation current responses 621, 623, 625, 627,629 and 631, which are generated in response to excitation pulses 521,523, 525, 527, 529 and 531, respectively. Excitation current responses621, 623, 625, 627, 629 and 631 include a Faradaic current responsecomponent relating to an electrochemical reaction in the test cell aswell as a capacitive charging current response relating to capacitiveelectrode charging and current response information attributable to aplurality of confounding variables. Current responses 622, 624, 626,628, 630 and 632 include a recovery current response relating todischarge of the test cell when maintained in a closed circuit conditionapplying a recovery potential and current response informationattributable to a plurality of confounding variables.

Current responses 621-632 include information related to theconcentration of an analyte of interest that may be present in thefluidic sample being tested, as well as additional information ofconfounding variables convolved therewith. This inventive conceptdescribed herein therefore can be incorporated into methods by which theinformation associated with current responses 621-631 can be used todetermine a concentration of an analyte of interest with enhancedaccuracy, precision, repeatability and reliability by compensating foror decreasing sensitivity to one or more confounding variables. A numberof confounding variables may impact analyte concentration determinationsincluding variations in reagent film thickness, sample temperature,sample Hct, reagent wetting, and reaction kinetics among others. Thepresent disclosure demonstrates that the methods disclosed herein may beutilized to perform analyte concentration determinations that compensatefor or exhibit decreased sensitivity to such confounding variables.

FIG. 6 shows in greater detail a portion 700 of the signals illustratedin FIGS. 4-5. The closed circuit recovery potential 522 ramps toexcitation potential 523 over a rate controlled ramp potential 752 suchas, for example, a ramp rate of about 45 mV/msec. Other embodimentscontrol the ramp rate between pulses at different rates that areeffective to reduce the contribution of the effect of capacitivecharging on current responses.

The ramping rate of ramp potential 752 is effective to reduce the effectof capacitive charging on current response 762, which is generated inresponse to ram p potential 752 and excitation potential 523. Current ismeasured starting about 30 msec after excitation potential 523 isachieved over an about 100 msec measurement period ending at the pointat which excitation potential 523 begins to ramp down to closed circuitrecovery potential 522 over ramp potential 753. Similar currentmeasurements may be taken for excitation current responses 621, 625,627, 629 and 631. It shall be appreciated that average currentmeasurements may be performed using continuous integration, discreteintegration, sampling or other averaging techniques. The successivecurrent measurements may be used to construct an effective current decaycurve from which analyte concentration can be calculated usingtechniques such as Cottrell analysis and others. In FIG. 6, ramppotential 752, ramp potential 753 is controlled to have a ramp ratesubstantially the same as ramp potential 752. In other instances, ramppotential 753 may be controlled at different rates or may be allowed totransition at a system defined rate without active control.

In connection with ramp rates, it can be seen in FIG. 6 that one mayobserve a high peak response when a potential is applied (see, theinitial current response 762 on that shoots above the graph as thepotential is ramped up to about +450 mV, as well as the current responseas the potential is ramped down to about 0 mV). To protect components ofthe measurement meter or system and/or to prevent interference with theelectrochemical reaction, it may be necessary to shunt the initiallyobserved current response and/or the lastly observed current response. Ashunting switch could be closed when sensor currents may exceed themaximum allowable input current to prevent an I-V amplifier fromsaturating. Alternatively, or in addition, it may be necessary toexclude the ramped parts of the current response and use responseinformation from a measurement window 750 as shown in FIG. 6.

Current responses, such as current responses 621-632, therefore encodeunique time ordered information relating to sample glucoseconcentration, sample hematocrit, sample temperature, as well asinformation relating to processes such as reagent wetting of thereagent, sample diffusion and separation with respect to the reagent,the establishment of a stable glucose transport mechanism, and thekinetics associated with the reducible analyte. Pulse sequences such aspulse sequence 520 provide short, distinct strobing of these processeswith respect to time and produces current responses including unique,time-ordered information relating to sample glucose concentration,sample Hct, sample temperature, and other factors. The inventors havedemonstrated a number of unexpected advantages of the methods disclosedherein through experiments in which pulse sequences such as pulsesequence 520 were used to analyze various concentrations of bloodglucose while Hct and temperature were varied systematically.

FIG. 7A shows another exemplary test sequence of (1) an AC block(labeled AC Block) including a plurality of low-amplitude signals; (2) aDC block (labeled DC Block 1) including short +450 mV pulses separatedby relaxation potentials at 0 mV, where the mediator is not oxidized bythe applied potential; and (3) a second DC block (labeled DC Block 2)including a slow-ramped, bi-polar potential (SRBP) at two different ramprates. More specifically, and as shown in FIG. 7B, the AC block includesfive (5) segments different at four (4) frequencies, namely 10 kHz, 20kHz, 10 kHz, 2 kHz and 1 kHz. Current response information to the ACblock may be used to determine admittance and phase values or othercomplex parameters as described in further detail below. In someinstances, an analyte concentration determination, such as a bGdetermination, is performed based upon current response information fromthe AC block and current response information from DC Block 1. Currentresponse information from DC Block 2, however, may be used forconstructing a glucose failsafe.

The second DC block shown in FIGS. 7A-B arose out of research relatingto SRBP excitation sequences. In theory, any DC excitation withsufficient potential to cause an electrochemical reaction of mediator onthe electrodes will produce a current response that can be used toquantitatively measure an analyte such as glucose. This current responsealso will be impacted by changing hematocrit and temperature levels.This research assessed the value of SRBP test sequences to determinewhether additional, unique information could be obtained and used toimprove analyte measurement system performance and/or capabilities, inmuch the same way that the use of recovery pulse information incombination with excitation pulse information can be used to improveperformance.

At the frequencies used, the current response to the first AC block inFIG. 7B does not contain information about glucose because the amplitudeof the signal is below the redox potential for the reagent-analytesystem, but it does encode information about Hct, temperature, and otherfactors, which can be used to correct a blood glucose (bG) readingderived from the DC test block(s). The current response to DC Block 1corresponds primarily to an amount of, for example, phenylenediamine(PDA; part of a known bG mediator system), which is proportional to theamount of glucose present. In contrast, the current response to DC Block2 provides quantitative information about levels of, for example,quinonediimine (QDI; also part of the bG mediator system), as well asPDA. Like DC Block 1, the current responses at +450 mV and −450 mVcorrespond to PDA, and are proportional to the amount of glucosepresent. However, the SRBP also enables the detection of QDI at lower,mid-range applied potentials during the negative- and positive-goingapplied potential ramps.

FIG. 8 shows the effects of an exemplary systematic variation ofexcitation current responses and recovery current responses to pulsesequence 520 described above for varying Hct and constant temperature.Current response sequences are illustrated for four test samples withvarying Hct concentrations of about 29.5%, 40.5%, 54% and 69.5%,constant glucose concentrations of about 530 mg/dL, and constanttemperatures of about 25° C. The magnitude and decay rates of theexcitation current responses to excitation potential pulses 521, 523,525, 527, 529 and 531 vary with sample Hct in a manner that issubstantially constant with respect to time. At each Hct, currentresponses 801, 803, 805, 807, 809 and 811 exhibit substantiallyconsistent magnitudes and decay rates for each pulse in pulse sequence520. Within each pulse of pulse sequence 520, the magnitude of currentresponses 801, 803, 805, 807, 809 and 811 varies in an inverserelationship with Hct.

The magnitude and growth rates of the recovery current responses torecovery potential pulses 522, 524, 526, 528, 530 and 532 also exhibitan observable relationship. Recovery current responses 802, 804, 806,808 and 810 to closed circuit recovery potential pulses 522, 524, 526,528, 530 and 532 have comparable starting magnitudes both within eachpulse and across pulses for each Hct, but have different rates of growthresulting in current response crossovers. As Hct varies, currentresponses 802, 804, 806, 808 and 810 grow at different rates dependingupon the Hct. The aforementioned current response characteristics andrelationships also were demonstrated in experiments that used sampleshaving constant glucose concentrations of about 33 mg/dL but wereotherwise substantially in accordance with those described above.

In comparison, FIG. 9 shows the effects an exemplary systematicvariation of current responses to pulse sequence 520 for varyingtemperature, constant Hct and constant glucose concentration. Currentresponses are illustrated for five test samples with varyingtemperatures of 6.5° C., 12.5° C., 24.6° C., 32.4° C. and 43.7° C.,constant Hct of about 41%, and constant glucose concentrations of about535 mg/dL. The current responses to the positive DC potential of pulses521, 523, 525, 527, 529 and 531 show a relative decrease for successivepulses with respect to time. The magnitude of current responses 901,903, 905, 907, 909 and 911 decrease successively across pulses for eachof the sample temperatures. Furthermore, the amount of decrease acrosspulses varies depending upon sample temperature.

The magnitude and growth rates of the recovery current responses 902,904, 906, 908 and 910 to recovery potential pulses 522, 524, 526, 528,530 and 532 also exhibit an observable relationship. Recovery currentresponses 902, 904, 906, 908 and 910 show substantially consistentmagnitudes across pulses and, within each pulse, have distinctly orderedstarting values and decreasing growth rates, but exhibit no crossover.The aforementioned current response characteristics and relationshipsalso were demonstrated in experiments that used samples having constantglucose concentrations of about 33 mg/dL but were otherwisesubstantially in accordance with those described above.

From this research, a number of current determination methods will nowbe described that use information from excitation current responses andclosed circuit recovery responses disclosed herein that compensate foror increase insensitivity to variation in sample temperature, sampleHct, or both. According to certain methods, a current value isdetermined from the current responses to pulsed sequences such as thosedescribed herein and is used to calculate analyte concentrationaccording to the Cottrell equation.

In one example, current was determined based upon a combination of thelast measurement point in one excitation current response and one closedcircuit recovery response. This current determination compensated forand exhibited decreased sensitivity to variation in sample temperatureand variation in sample Hct relative to current determinations basedupon only excitation current response information.

Likewise, the measurement methods can be used to compensate for orincrease insensitivity to variation in reagent thickness or to increasedanalyte measurement insensitivity to variation in reagent thickness.Exemplary methods are demonstrated in a series of experiments, theresults of which are shown in FIGS. 10-14. The experiments measuredworking electrode current responses to a test signal including asequence of DC excitation pulses and closed circuit recovery pulsessimilar to those disclosed herein above for varying reagent filmthicknesses.

FIG. 10 shows current responses to a DC pulse sequence in which aworking electrode potential was ramped between excitation potentialpulses about +450 mV in amplitude and about 130 msec in duration andclosed circuit recovery potential pulses about 0 mV in amplitude andabout 280 msec in duration. Current response measurements were takenduring the last 100 msec of each excitation potential pulse. Currentresponses 1410-1416 represent an average current measured during thelast 100 msec of the excitation potential pulses applied to a testelement with a reagent film thickness of about 4.4 μm was provided byapplication of a wet coat weight of about 45 g/m². Current responses1420-1426 represent an average current measured during the last 100 msecof the excitation potential pulses applied to a biosensor with a reagentfilm thickness of about 5.5 μm was provided by application of a wet coatweight of about 5.5 g/m².

FIG. 10 further shows current deltas (Δ) 1430-1436, which denote thepercent delta between current response measurements 1410-1416 andcorresponding current measurements 1420-1426. As shown by currentresponse 1434, a current response delta of about 5.2% was achieved atabout 3.7 sec after sample detection. In contrast, the correspondingcurrent response deltas for a constant DC potential of about 450 mVapplied before and maintained after sample dosing remained above about8% as late as 5 sec after sample detection. Similarly, the currentresponse deltas for a stepped DC potential of about 450 mV applied about3 sec after sample dosing and maintained thereafter remained above about8% as late as 5 sec after sample detection.

FIG. 11 shows current responses to a DC pulse sequence similar to thatof FIG. 10, but for ten (10) excitation potential pulses appliedstarting immediately after a sample is detected. Current responses1510-1519 represent an average current measured during the last 100 msecof the excitation potential pulses applied to a test element with areagent film thickness of about 4.4 μm. Current responses 1520-1529represent an average current measured during the last 100 msec of theexcitation potential pulses applied to a test element with a reagentfilm thickness of about 5.5 μm. Current deltas 1530-1539 illustrate thatthe current deltas due to reagent film thickness are initiallycomparable to those of FIG. 10, but decrease to about 3.9% at about 3.7sec after sample detection.

FIG. 12 shows a test sequence in which a preconditioning potential 1610of about 450 mV and about 500 msec in duration was applied to a workingelectrode at sample detection. About 1 sec after sample detection, a DCpulse sequence 1620 was applied in which working electrode potential wasramped between excitation potential pulses about 450 mV in amplitude andabout 130 msec in duration and closed circuit recovery potential pulsesabout 0 mV in amplitude and about 280 msec in duration. FIG. 12 furthershows current deltas 1630-1635, which indicate the difference betweenthe average current measured during the last 100 msec of the excitationpotential pulses for reagent film thicknesses of about 4.4 μm and 5.5μm. Here, a current response delta of about 2% was achieved at about 3.7sec after sample detection.

Further experimentation involving preconditioning pulses similar to thatused in connection with FIG. 12 revealed that pulses with the samepolarity as subsequent excitation pulses were effective to decreasecurrent measurement deltas for variations in reagent thickness. Thisexperimentation also revealed that the reduction in such current deltasincreased as the magnitude of the preconditioning pulses increased.

FIG. 13 shows a test sequence in which a two-cycle triangularpreconditioning waveform 171 that alternates between a potential of −450mV and +450 mV and is about 1800 msec in duration and that was appliedto a working electrode about sample detection. Starting about 200 msecafter sample detection, a DC pulse sequence 172 was applied, in whichworking electrode potential was ramped between excitation potentialpulses about +450 mV in amplitude and about 130 msec in duration andclosed circuit recovery potential pulses about 0 mV in amplitude andabout 280 msec in duration.

FIG. 14 illustrates a graph of current responses and current responsedeltas corresponding to the test sequence of FIG. 13. Current responses1710-1715 represent an average current measured during the last 100 msecof the excitation potential pulses applied to a test element with areagent film thickness of about 4.4 μm. Current responses 1720-1725represent an average current measured during the last 100 msec of theexcitation potential pulses applied to a test element with a reagentfilm thickness of about 5.5 μm. Current deltas 1730-1735 illustrate thatthe current deltas due to dry film thickness are initially lower thanthose of FIGS. 10-11 and decrease to about 0.8% about 3.7 sec aftersample detection.

All of the patents, patent applications, patent application publicationsand other publications recited herein are hereby incorporated byreference as if set forth in their entirety.

The present invention has been described in connection with what arepresently considered to be the most practical and preferred embodiments.However, the invention has been presented by way of illustration and isnot intended to be limited to the disclosed embodiments. Accordingly,one of skill in the art will realize that the invention is intended toencompass all modifications and alternative arrangements within thespirit and scope of the invention as set forth in the appended claims.

What is claimed is:
 1. A method for electrochemical measurement of ananalyte in a fluid sample comprising: applying an electrical testsequence to an electrochemical biosensor, the electrochemical biosensorcomprising: an electrode system; a reagent in electrical communicationwith the electrode system; a receptacle configured to place the fluidsample provided to the electrochemical biosensor in fluidic contact withthe reagent; and the electrical test sequence further comprising:applying a preconditioning signal to the electrode system; applying atleast two direct current (DC) excitation signals to the electrode systemafter the applying of the preconditioning signal; and applying at leastone recovery potential to the electrode system by maintaining a closedcircuit condition of the electrode system, each recovery potential inthe at least one recovery potential being generated between two DCexcitation signals in the at least two DC excitation signals; generatinga current measurement from the electrode system during at least one DCexcitation signal in the at least two DC excitation signals of theelectrical test sequence; and determining a concentration of an analytein the fluid sample based at least in part on the current measurement.2. The method of claim 1, wherein the preconditioning signal is anotherDC excitation signal.
 3. The method of claim 2, wherein thepreconditioning signal has a first duration that exceeds a secondduration of each DC excitation signal in the at least two DC excitationsignals.
 4. The method of claim 3, wherein the DC excitation of thepreconditioning signal has a duration of about 500 milliseconds and eachDC excitation signal in the at least two DC excitation signals has aduration of about 130 milliseconds.
 5. The method of claim 2, whereinthe current measurement has a deviation of less than 3% for anythickness of the reagent in a range of about 4.4 μm to about 5.5 μm fora single analyte concentration of the fluid sample.
 6. The method ofclaim 1, wherein the preconditioning signal is an alternating current(AC) excitation signal.
 7. The method of claim 5, wherein the ACexcitation signal is generated with a triangular waveform.
 8. The methodof claim 7, wherein the triangular waveform is a two-cycle triangularwaveform that alternates between a potential of −450 mV and +450 mV andis about 1800 msec in duration.
 9. The method of claim 8, wherein eachDC excitation signal in the at least two DC excitation signals has aduration of about 130 milliseconds and an amplitude of about +450 my.10. The method of claim 6, wherein the current measurement has adeviation of less than 1% for any thickness of the reagent in a range ofabout 4.4 μm to about 5.5 μm for a single analyte concentration of thefluid sample.
 11. The method of claim 1, wherein each DC excitationsignal is generated with an amplitude of about +450 mV and each recoverysignal is generated with an amplitude of about 0 mV, and wherein each DCexcitation signal and each recovery signal is applied for a time periodin a range of approximately 50 msec to 500 msec.
 12. The method of claim1 wherein the fluid sample is blood and the analyte is glucose.